Occurrence and mechanisms of tigecycline resistance in carbapenem- and colistin-resistant Klebsiella pneumoniae in Thailand

Tigecycline has been regarded as one of the most important last-resort antibiotics for the treatment of infections caused by extensively drug-resistant (XDR) bacteria, particularly carbapenem- and colistin-resistant Klebsiella pneumoniae (C-C-RKP). However, reports on tigecycline resistance have been growing. Overall, ~ 4000 K. pneumoniae clinical isolates were collected over a five-year period (2017–2021), in which 240 isolates of C-C-RKP were investigated. Most of these isolates (91.7%) were resistant to tigecycline. Notably, a high-risk clone of ST16 was predominantly identified, which was associated with the co-harboring of blaNDM-1 and blaOXA-232 genes. Their major mechanism of tigecycline resistance was the overexpression of efflux pump acrB gene and its regulator RamA, which was caused by mutations in RamR (M184V, Y59C, I141T, A28T, C99/C100 insertion), in RamR binding site (PI) of ramA gene (C139T), in MarR (S82G), and/or in AcrR (L154R, R13Q). Interestingly, four isolates of ST147 carried the mutated tet(A) efflux pump gene. To our knowledge, this is the first report on the prevalence and mechanisms of tigecycline resistance in C-C-RKP isolated from Thailand. The high incidence of tigecycline resistance observed among C-C-RKP in this study reflects an ongoing evolution of XDR bacteria against the last-resort antibiotics, which demands urgent action.

The expression level of AcrAB-TolC efflux pump and the regulator RamA among efflux pump positive isolates were evaluated using qRT-PCR.As compared to K. pneumoniae ATCC 13883, the result revealed an alteration in expression level of the efflux pump (acrB) and the regulator (ramA) (Table S2).Among the high-level tigecyclineresistant group, most isolates showed upregulation of acrB (1.2 to 8.1-fold) and upregulation of ramA (1.9 to 24.9-fold).Conversely, in the low-level tigecycline-resistant group, expression of acrB was slightly increased (1.01 to 2.63-fold) while expression level of ramA was diverse (1.50 to 9.83-fold).
The expression levels of acrB and ramA between the two tigecycline-resistant groups were evaluated using Mann-Whitney U test.The result showed that acrB expression level was significantly increased in high-level tigecycline-resistant group when compared to that of low-level tigecycline-resistant group (P = 0.0002) (Fig. 2a).However, the expression level of ramA showed no significant difference between these two groups (P = 0.3698) (Fig. 2b).

Whole genome sequence (WGS) analysis of tigecycline resistant isolates
To identify the causes of tigecycline resistance in C-C-RKP, 15 isolates with positive efflux pump activity were selected and subjected to whole genome sequencing.The analysis focused on mutations in the regions which have been reported to have a correlation with the overexpression of AcrAB-TolC efflux pump, including RamR (ramA repressor), PI and PII regions (recognition sites of RamR in romA-ramA operon), AcrR (acrAB repressor), and Lon protease (ramA regulator).Mutations within repressors of the other AcrAB-TolC regulators including MarA and SoxS were also considered.Apart from AcrAB-TolC efflux pump, other mechanisms reported to confer tigecycline resistance including mutations in rpsJ gene (a ribosomal S10 encoding gene), mutation in OqxR (OqxAB efflux pump regulator), acquisition of Tet(A) efflux pump, TmexCD1-ToprJ1 efflux pump, and Tet(X) (a tigecycline degrading enzyme) were investigated.WGS analysis revealed that most of the isolates contained mutations within RamR except for XDR-KP-047 and XDR-KP-059 (Table 2).Among high-level tigecyclineresistant isolates, three major patterns of RamR mutations were observed.First, a certain number of isolates were presented with a C-nucleotide insertion in ramR gene at position 99 or 100, leading to a frameshift mutation causing premature stop codon at amino acid position 53.These isolates also possessed C139T point mutation in RamR recognition site PI, which might prevent RamR binding leading to the overexpression of ramA and acrB.Second, certain isolates had S157P mutation in RamR without additional mutations in either RamR recognition site PI or AcrR.Third, a group of isolates possessed co-mutations of RamR and RamR recognition site PI, and/or AcrR.For instance, XDR-KP-113 contained double mutations in RamR (A28T and I141T) and RamR recognition site PI (C139T), XDR-KP-051 carried a single point mutation within both RamR (A20D) and AcrR (W50R), and XDR-KP-206 possessed co-mutation within these three regions.
Among low-level tigecycline-resistant isolates, most of which also possessed mutations in RamR, and RamR recognition site PI.However, most of these mutations, especially I141T and A19V in RamR and R13W in AcrR, have been reported to have no effects on overexpression of RamA.In addition, mutations within MarR and SoxR, the repressor of MarA and SoxS, respectively, were investigated.Within MarR, as compared to K. pneumoniae ATCC 13883, a single point mutation S82G was detected in all isolates and double mutations S3N/S82G were detected in 3 isolates of high-level tigecycline-resistant group.No mutations were detected for SoxR.Apart from an overexpression of AcrAB-TolC efflux pump, four isolates were found to harbor Tet(A) efflux pump.All of them were the mutated Tet(A) protein, which contained 7 mutations including I5R, V55M, I75V, T84A, S201A, F202S, V203F.Two of the four isolates, XDR-KP-011 and XDR-KP-047, also possessed overexpression of acrB (Table 2).  . No.

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no. www.nature.com/scientificreports/Thus, co-existence of the mutated Tet(A) efflux pump and overexpression of acrB were believed to be the cause of high-level tigecycline resistance (TGC MIC 8 µg/ml) in these two isolates.For the other 2 isolates, XDR-KP-059 and XDR-KP-072, Tet(A) appeared to be solely responsible for tigecycline resistance since the expression of acrB was only 1.5-fold upregulated.Mutations in rpsJ and OqxR repressor were not found and mobilized tigecycline resistance genes including Tet(X) and TmexCD-ToprJ efflux pump were not detected.

Clonal relatedness among tigecycline resistant C-C-RKP
The isolates showing tigecycline MIC ranging from 4 to 8 µg/ml were selected to perform ERIC-PCR.According to the result, similarity of genotypic fingerprints among these isolates was ranging from 51.4 to 100%.Clusters were differentiated using 85% similarity, and ERIC types were classified using 95% similarity.Twenty-four clusters were distinguished (cluster A to X) and two major clusters including cluster I (14%) and W (12%) were identified (Fig. 1).Among these clusters, 51 ERIC types were differentiated.).These isolates also showed high-level resistance to tigecycline (MIC 4 µg/ml) with an activity of efflux pump.In addition, these isolates possessed a major frameshift mutation in ramR and a mutation in RamR recognition site PI, resulting in an overexpression of both ramA and acrB.These results suggested that the clonal dissemination of chromosomal AcrAB-TolC mediated tigecycline resistance was found among some isolates.However, the dissemination of acquired resistance genes including carbapenemase and tet(A) genes using the horizontal gene transfer were detected since same genes were found among the different clusters, especially for tet(A), which was identified from different ERIC types.
According to the MLST analysis, five STs including ST15, ST16, ST101, ST147, and ST231 were identified.The major STs among tigecycline-resistant C-C-RKP isolates were ST16 and ST147.In ST16, most of the isolates were associated with co-harboring of bla NDM and bla OXA-48-like genes.For tigecycline resistance, overexpression of acrB and ramA appeared to be the main mechanism, which was caused by the co-mutations of ramR gene, RamR recognition site (PI promoter) and/or acrR gene.In ST147, four isolates were identified and most of them harbored only bla NDM gene.Interestingly, all isolates were found to harbor the mutated tet(A) gene which encoded 7 mutations including I5R, V55M, I75V, T84A, S201A, F202S, V203F, compared to wild-type Tet(A) (GeneBank accession number X00006.1).Based on cgSNP, phylogenetic analysis of all published genomes of tigecycline-resistant K. pneumoniae isolates from more than nine countries across the world (15 from this study and 76 from NCBI database) revealed eight major clades (Fig. 3).The MIC levels and mechanisms of tigecycline resistance appeared to be diverse.Notably, the co-existence of AcrAB-TolC overexpression and Tet(A) efflux pump consistently conferred high-level of resistance.The C-C-RKP isolates in our study were classified into 4 clades, including clade 1, 2, 4, and 7.In clade 1, C-C-RKP ST101 and ST231 were closely related to K. pneumoniae ST383 clinically isolated from Lebanon 15 .This isolate showed pan drug-resistant (PDR) phenotype, which harbored 47 AMR determinants including carbapenemase genes (bla NDM-5 and bla OXA-48 ), chromosomal-mediated colistin resistance, as well as tigecycline resistance due to mutations in RamR and Tet(A) efflux pump (X61367).All members of clade 2 belonged to clonal group 15, which included OXA-232-producing C-C-RKP ST14 isolates from South Korea 16 , and K. pneumoniae ST15 isolates from the United States 17 and China 18 .Two C-C-RKP ST15 from our study showed close association with clinical isolates from the USA 17 and China 18 .The isolate from the USA showed PDR phenotype which harbored bla NDM-1 for carbapenem resistance, disrupted mgrB gene for colistin resistance, mutated ramR and mutated Tet(A) efflux pump (GeneBank accession number X61367.1) for tigecycline resistance.An additional ST15 strain from China was KPC-2-producing K. pneumoniae which was resistant to almost all tested antibiotics except for colistin.This isolate showed high-level tigecycline resistance which was caused by mutations in ramR and acquisition of tet(A).Clade 4 consisted of our ST16 isolates and K. pneumoniae ST17 isolates from China 19 .Lastly, clade 7 comprised all of our ST147, which showed close relationship with a NDM-5 and OXA-181 co-producing K. pneumoniae ST147 from South Korea 20 .In contrast to our ST147 isolates, the Korean isolate did not carry a mutated Tet(A) efflux pump.Instead, it exhibited tigecycline resistance due to the insertion of a DNA fragment flanking the RamR recognition site within the romA gene.Furthermore, our ST147 isolates shared the same origin with KPC-2 and NDM-1 co-producing K. pneumoniae ST464, which also harbored mutated Tet(A) efflux 21 .

Discussion
The emergence of carbapenem and colistin resistance in K. pneumoniae has intensified the antimicrobial resistance problem since few treatment options are left available.Tigecycline is one of the last-resort drugs that has been recommended for treatment of C-C-RKP infection 5 .Nevertheless, tigecycline resistance has been increasingly reported.In this study, a high rate of tigecycline resistance (91.7%) was observed among C-C-RKP, consistent with recent findings in Thailand that tigecycline resistance rate in carbapenem-resistant K. pneumoniae ranged from 46.7 to 79.6% [22][23][24] .However, it is worth noting that a clinical breakpoint of tigecycline resistance for Klebsiella spp. is not yet available in CLSI and EUCAST interpretive guidelines.Thus, tigecycline resistance rates in these studies were reported based on EUCAST breakpoints recommended for E. coli and C. koseri (MIC > 0.5 µg/ml) 25 .In recent years, it has been demonstrated that the dosage regimen of tigecycline could reach the serum concentrations of up to 2 µg/ml 26 and several surveillance programs have reported tigecycline MIC 90 at approximately 2 µg/ml among Klebsiella isolates 27,28 .Therefore, in accordance with these studies, the isolate with tigecycline MIC greater than 2 µg/ml were considered by US-FDA 29 and BSAC 30 as a non-susceptible and a resistant isolate, respectively.Interestingly, although 2 µg/ml was applied, tigecycline resistance rate among C-C-RKP in our study was still high (23.8%).This rate was even higher than that of the other global reports in carbapenem-resistant isolates, ranging from 1.9 to 8.9% [31][32][33][34][35] .
Apart from carbapenems, colistin, and tigecycline, the isolates in our study also showed high rate of resistance to several antibiotics.However, up to 70% of the isolates were still susceptible to gentamicin and chloramphenicol, coherent with the report of the National Antimicrobial Resistance Surveillance Center Thailand (NARST) in 2022 36 and with the other study 37 that the susceptibility rates of gentamicin and chloramphenicol among K. pneumoniae were higher than 80%.Moreover, several studies has highlighted the potential of these two agents as treatment options against multidrug-resistant K. pneumoniae (MDR-KP) 22,38 .Therefore, gentamicin and chloramphenicol seem to be the most effective agents against MDR-KP, in vitro.Treatment of infection using these drugs in combination with β-lactams/β-lactamase inhibitor reserved for MDR bacteria such as ceftazidime-avibactam, meropenem-vaborbactam, and imipenem-relebactam may provide the best treatment outcome 6 .
Characterization of carbapenem-resistance mechanisms in this study revealed that co-carriage of bla NDM and bla OXA-48-like genes was the most prevalent genotype, followed by bla NDM gene, and bla OXA-48-like gene, respectively.However, other carbapenemase genes including bla IMP , bla VIM , and bla KPC were not detected.These findings are in an accord with the results from the previous studies that the prevalence of K. pneumoniae carrying bla NDM and/ or bla OXA-48-like genes in Thailand is much higher than that of bla IMP , bla VIM and bla KPC genes [39][40][41][42] .Noteworthy, bla KPC genes in Thailand is extremely rare.
In this study, acrB overexpression was found in most tigecycline resistant isolates, most of which were associated with the upregulation of ramA.This is in accord with the previous studies.Tigecycline resistance in K. pneumoniae has been reported to associate with chromosomal-mediated overexpression of AcrAB-TolC efflux pump 10,[43][44][45][46][47] .Expression of the efflux pump genes (acrA and acrB) has been found to significantly increase in the resistant isolates 43,45,47,48 .In addition, linear correlation between expression level of acrB and tigecycline MIC has been reported 43,45 .Generally, the expression of the efflux pump genes is controlled by their local repressor AcrR, and the global activators such as RamA, MarA, and SoxS 10,49 .Therefore, the upregulation of the efflux pump genes has been frequently reported with overexpression of these regulators, especially RamA 12,43,50,51 .
Overexpression of RamA has been reported to be caused by several mechanisms, including dysfunctional mutations in RamR, mutations in RamR binding site within RamA promoter regions, and mutations of Lon protease.ramR is a transcriptional repressor gene belonging to the TetR family.It encoded the RamR repressor, www.nature.com/scientificreports/which binds to the promoter regions of the ramA gene, resulting in transcriptional repression.RamR protein consists of 194 amino acids forming 9 alpha helices protein which separated into N-terminal DNA binding domain (α1 to α3), and C-terminal dimerization domain (α4 to α9) 52 .The loss-of-function mutations in RamR have been reported from several species especially K. pneumoniae, which resulted in ramA overexpression 12,53,54 .In addition, computational analyses have indicated that mutations in the dimerization domain of RamR can lead to a reduction in its DNA binding affinity in Salmonella spp. 55.In our study, mutations in RamR were detected in both DNA binding and dimerization domains but mostly were point mutations that occurred outside hotspot regions.A frameshift mutation from a single nucleotide insertion was detected in two isolates.Most of the isolates with RamR mutations showed overexpression of RamA.In addition, mutations that have been associated with an inability to induce RamA upregulation, including A19V and I141T were detected 12,43,44,51,53 .Apart from RamR, in this study, the mutation within RamR recognition site was also identified.RamR binds to two regions upfront of ramA gene (PI and PII promoters) for regulating activity 51 .Yet only a mutation within the PI promoter region was detected.Notably, a C139T point mutation was identified in most of our isolates, which suggests the presence of a potential hotspot within the PI promoter region.Mutations in Lon protease that has been reported to associate with RamA upregulation were not detected 56 .Nevertheless, there were some isolates that showed no correlation between these two genes; upregulation of ramA was found without overexpression of acrB.To explain this discrepancy, further investigations are required.Noteworthy, one isolate with high-level tigecycline resistance, XDR-KP-185 (MIC 4 µg/ml), showed only 1.28-fold of acrB expression level while knownacquired resistant determinants were not detected.This might indicate an activity of other mechanisms, such as OqxAB or KpgABC efflux pump, which have also been reported to confer tigecycline resistance 50,57,58 .Since tigecycline-resistance mechanisms are complex and may involve several efflux pumps and regulators, enhancing the sensitivity of an efflux pump inhibition assay may require the use of multiple types of efflux pump inhibitors.
In addition, it may be beneficial to measure the expression levels of other efflux pumps, such as OqxAB, and the expression levels of other global regulators such as MarA, SoxS, and RarA.This study identified several important STs among XDR-K.pneumoniae isolates, including ST15, ST16, ST101, ST147, and ST231.Also, this was the first characterization of tigecycline-resistance mechanism from these STs in Thailand.In this work, ST16 was identified as the predominant clone.ST16 is globally known for its highrisk profile associated with both hypervirulent and drug-resistant characteristics.To date, the emergence of K. pneumoniae ST16 has been reported from several countries, including Denmark 59,60 , France 61 , Italy 62 , United Kingdom 63 , Ireland 63 , Croatia 64 , Bulgaria 65 , Canada 66 , Thailand 67,68 , Vietnam 69 , China 70 , Egypt 71 , and Brazil 72 , which covered 4 of 7 continents.Interestingly, very few studies demonstrated the association between tigecycline resistance and this high-risk clone.Nevertheless, K. pneumoniae ST16 has been reported to accumulate various types of antimicrobial resistance determinants 73 such as bla CTX-M-15 61 , bla KPC 74 , bla NDM-5 60 , bla OXA-48-like 75 , bla NDM-1 and bla OXA-232 62,67,68 , aac(6′)-lb-cr 64 , aac(6′)-lb 67 , qnrS 67 , and mgrB mutation 67,69 .In our study, the accumulation of many antimicrobial resistance determinants (bla CTX-M-15 , bla NDM-1 , bla OXA-232 , aadA1, aadA2, aac(6′)-lb-cr, and aac(6′)-lb) was also detected in C-C-RKP ST16 isolates.To the best of our knowledge, this study was the first to demonstrate the connection between the clonal expansion of K. pneumoniae ST16 and a high rate of tigecycline resistance among C-C-RKP isolates.Therefore, our finding could provide potential evidence to support the ongoing evolution of the K. pneumoniae ST16 against the last-resort antibiotics, indicating the capability of this high-risk clone to pose a significant public health threat.Another significant ST was ST147, recognized as a successful high-risk clone within K. pneumoniae which was reported to be associated with β-lactams and fluoroquinolones resistance 76 .ST147 has been reported globally, with multiple outbreaks associated with this ST, except in Antarctica 4 .It has been found to harbor various antimicrobial resistance genes, such as extendedspectrum β-lactamase, carbapenemase, aminoglycosides resistance, fluoroquinolone resistance, and tet(A) gene.In Thailand, ST147 carrying bla NDM have been reported in several studies 24,77 .However, the major clonal group of K. pneumoniae-CG258, comprising ST11, ST258, and ST512, was not detected in our study 4 .
The only acquired tigecycline-resistance mechanism detected in our study was the Tet(A) efflux pump.Four isolates of ST147 were found to harbor the same mutated Tet(A), which contained 7 mutations (I5R, V55M, I75V, T84A, S201A, F202S, V203F).This type of mutated Tet(A) was similar to that of the previous reports from China and Taiwan 12,21,78 .The mutated Tet(A) has been proved to confer tigecycline resistance via gene cloning experiment, which elevated tigecycline MIC of the susceptible organism up to 8-fold 12,78 .The gene has been reported to be located on plasmids 21,78 , suggesting a possibility of conjugative transfer of this gene to different bacterial species.Moreover, the co-existence of this mutated efflux pump with dysfunctional RamR has been reported to exacerbate tigecycline-resistance level 12 .In our study, the mutated Tet(A) was found to co-exist with acrB overexpression, which was found to confer high-level tigecycline resistance (MIC 8 µg/ml) in two isolates.This result suggests that beside dysfunctional RamR, the co-occurrence of the mutated Tet(A) with other resistance mechanisms may also be able to confer high-level tigecycline resistance.To our knowledge, this is the first report of mutated Tet(A) harboring C-C-RKP in Thailand.Therefore, presence of the mutated Tet(A) should be carefully monitored.Interestingly, we found that an efflux pump inhibitor CCCP could re-sensitize the isolates to tigecycline that harbor only Tet(A) resistance mechanism.The presence of 20 µg/ml CCCP could reduce the MIC of tigecycline from 2 and 4 µg/ml to 0.125 µg/ml in XDR-KP-059 and XDR-KP-072, respectively.This re-sensitization of isolates to tigecycline indicates effectiveness of an efflux pump inhibitor which could be a promising choice to combat Tet(A) harboring K. pneumoniae.

Bacterial strains
A total of 4179 K. pneumoniae clinical isolates were collected from public hospitals including King Chulalongkorn Memorial Hospital, Chulalongkorn University, and the Golden Jubilee Medical Center, Mahidol University,

Efflux pump inhibition assay
Efflux pump activity was assayed using efflux pump inhibitor, carbonyl cyanide m-chlorophenylhydrazone (CCCP) (TCI, Japan).Briefly, MIC of tigecycline was evaluated using agar dilution method in the presence and absence of 20 µg/ml of CCCP.Then, a 4-fold or greater reduction of tigecycline MIC observed in the presence of CCCP, compared to the absence of CCCP, was considered as positive for efflux pump activity 88 .

Measurement of AcrAB efflux pump expression level
Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was used to assess the expression levels of efflux pump gene acrB, and transcription activator gene ramA.The expression level of rrsE housekeeping gene was also measured and served as the reference for normalizing the expression level of each target gene.The specific primers were listed in Table S1 43 .Total bacterial RNA was extracted from log-phase culture using TRNzol Universal Reagent (Tiangen, China), and was treated with DNase I (ThermoFisher Scientific, Waltham, USA).Then, qRT-PCR was performed with KAPA SYBR ® FAST One-Step qRT-PCR (KAPA BIOSYSTEMS (PTY) LTD, South Africa).The final 15 µl PCR reaction included 30 ng of DNase treated RNA, 0.4 µM of primers for acrB reaction and 0.15 µM of primers for ramA and rrsE reactions.The PCR condition included cDNA synthesis at 42 °C for 15 min, pre-denaturation at 95 °C for 5 min, and 40 cycles of denaturation at 95 °C for 15 s and annealing at 60 °C for 60 s.The result was analyzed using CFX Maestro software based on the 2 −ΔΔct method 43 .Relative expression level of the mRNA was compared to that of K. pneumoniae ATCC 13883, a tigecycline-susceptible reference strain.

Whole genome sequencing
Single colony of the selected C-C-RKP isolates with tigecycline-resistant phenotype was cultured overnight.Then, gDNA was extracted and purified using DNeasy ® Blood and Tissue Kit (QIAGEN, Germany).The library preparation was performed using VAHTS Univeral DNA Library Prep kit (Vazyme Biotech, China).The gDNA was sequenced using Illumina Novaseq 6000, PE150 platform (Illumina, San Diego, CA, USA).The quality of raw read was investigated using FastQC tool (Version 0.12.1) 89.Then, raw data was trimmed using TrimGalore (version 0.6.10) 90.Bacterial genomes were later assembled using Spades (version 1.1.0)and Quast tool (Version 5.2.0) was performed to assess quality of the assembly 91,92 .Prokka (version 1.14.6) was used to annotate genes 93 .The antimicrobial resistance and plasmid were determined by Staramr tool (version 0.9.1) which cooperated with ResFinder, and PlasmidFinder, respectively 94 .The genomes were compared using the Basic Local Alignment Search Tool (BLAST, NCBI) 95 .The analysis of gene mutation was based on a comparison of the C-C-RKP′ Vol:.( 1234567890

Phylogenetic analysis
Core genome single nucleotide polymorphisms (cgSNP) was performed to determine the number of core genome SNP from draft genomes of tigecycline-resistant C-C-RKP clinical isolates in Thailand and from varying tigecycline-resistant K. pneumoniae WGS investigations conducted elsewhere (available on NCBI database).K. pneumoniae subsp.pneumoniae MGH 78578 (NCBI RefSeq assembly GCF_000016305.1) was used as a reference to generate a core genome alignment and phylogenetic tree was constructed using a core SNP alignment.Draft genomes of K. pneumoniae were aligned following the detection and filtration of recombinant regions using Parsnp v1.2 96 and Gubbins v2.4.1 97 .Maximum-likelihood (ML) trees were generated by RAxML v8.2.12 98 using ASC_GTRGAMMA model of rate heterogeneity with the Lewis correction for ascertainment bias 99,100 .Branch support was performed by 1000 bootstrap replicates.Best-scoring ML tree was visualized and annotated as a phylogenetic tree using FigTree v1.4.4 and Evolview v2 101,102 .

Statistical analysis
Statistical analysis of an association between tigecycline-resistance level and expression level of acrB and ramA was performed using GraphPad Prism 8 software.Since the data were not normally distributed, an intergroup comparison was performed using the Mann-Whitney U test.A P value < 0.05 was considered as statistical significance.The correlation between expression level of acrB and tigecycline MIC was evaluated using linear regression. Vol

Figure 1 .
Figure 1.Dendrogram based on ERIC-PCR typing, antimicrobial susceptibility profile, resistance gene profile, and efflux pump activity of 57 C-C-RKP isolates with tigecycline MIC ranged from 4 to 8 µg/ml.The black, gray, and white color in antimicrobial susceptibility profile indicate resistant, intermediate, and susceptible phenotype to an antibiotic, respectively.a Fold reduction of tigecycline MIC in presence of 20 µg/ml CCCP compared to absence of CCCP.b Screening of tigecycline resistant gene including tet(A), tet(X), and tmexCD1-toprJ1.

Figure 3 .
Figure 3. Phylogenetic analysis based on core genome single nucleotide polymorphisms (cgSNP) of 15 C-C-RKP isolates from this study (red labeled) and 76 isolates of tigecycline-resistant K. pneumoniae retrieved from NCBI database.